Patent Application
20210163947
A sequence listing, provided as an ASCII text file and submitted via EFS-Web as part of the present specification was created on Dec. 8, 2020 is named 702581_01883_ST25.txt, is 168 KB, and is incorporated herein by reference in its entirety.
The field of the invention relates to cell-free protein synthesis (CFPS) systems. In particular, the field of the invention relates to the use of CFPS systems for in vitro detection of target molecules using cellular extracts.
Cell-free systems offer practical and technical advantages over whole-cell sensors for point-of-use detection of contaminants in aqueous environments like lead, arsenic, mercury, fluoride, and nitrate, and for detecting chemical markers of health and performance in human samples such as blood, urine and saliva. However, the diversity of sensors that can function in E. coli extracts is constrained by the scarcity of characterized strong promoters that can be regulated by allosteric transcription factors. Because engineering promoter strength without affecting inducibility remains an unsolved challenge in synthetic biology, the output signals from cell-free sensors are often undesirably low, particularly when detecting trace contaminants.
To address this problem, here we disclose a platform that utilizes CFPS for in vitro sensing of metabolites including small-molecule metabolites in which the output from a cell-free sensor is amplified using an intermediate RNA polymerase synthesized in situ. Positive feedback introduced through autocatalytic transcription and translation decreases the time required for a generating a detectable signal. By employing orthogonal polymerases in parallel, multiple key target chemicals can be detectable simultaneously in a single reaction vessel. The disclosed technology will have transformative impact toward the engineering of highly sensitive and field-deployable cell-free biosensors for monitoring metabolites and contaminants and may have wide applications including applications for monitoring global water quality.
Disclosed are methods, devices, kits, components, and compositions for detecting a target molecule in a test sample using a cell-free protein synthesis (CFPS) reaction. The methods, devices, kits, components, and compositions may be utilized for detecting target molecules which may include small molecules and/or metabolites of small molecules. The components used in the disclosed methods, devices, and kits may be dried or lyophilized and may be present or immobilized on a paper substrate.
The disclosed methods, devices, kits, components, and compositions typically utilize one or more transcription templates that encode and conditionally express one or more exogenous RNA polymerases in the presence of the target molecule. The expressed RNA polymerases in turn induce expression of one or more reporter molecules from transcription templates comprising promoters for the RNA polymerases, thereby amplifying an output signal that is generated in the presence of a detected target molecule.
The disclosed methods may be performed to detect a target molecule in a biological or environmental sample and may include steps of: (i) obtaining a biological or environmental sample which may or may not contain the target molecule and optionally concentrating and/or solubilizing the target molecule in the sample if necessary; and (ii) adding the sample and/or the optionally concentrated and/or solubilized target molecule in the sample to a cell-free protein synthesis (CFPS) reaction, where, if the target molecule is present in the sample, then an output is generated and amplified using an intermediate RNA polymerase synthesized in situ. The disclosed methods utilized positive autocatalytic transcription and translation which decreases the time required for generating a detectable signal.
In some embodiments, the disclosed compositions, kits, systems, or methods include an inhibition scheme to minimize background production, in the absence of the target molecule, of one or more RNA polymerases employed in the compositions, kits, systems, or methods. In some embodiments, the inhibition scheme comprises an inhibitor, optionally wherein the inhibitor is selected from a T7 lysozyme, an RNA or DNA aptamer against T7 RNAP, a DNA mimic of the native T7 RNAP promoter recognition sequence, a sequence-responsive protease that selectively degrades tagged T7 RNAP, and combinations thereof. In some embodiments, the inhibitor comprises a protease, such as basal ClpX protein.
The presently disclosed subject matter is described herein using several definitions, as set forth below and throughout the application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a component,” “a metabolite,” and “a contaminant,” should be interpreted to mean “one or more components,” “one or more metabolites,” and “one or more contaminants,” respectively. For example, “a composition,” “a system,” “a kit,” “a method,” “a protein,” “a vector,” “a domain,” “a binding site,” and “an RNA” should be interpreted to mean “one or more compositions,” “one or more systems,” “one or more kits,” “one or more methods,” “one or more proteins,” “one or more vectors,” “one or more domains,” “one or more binding sites,” and “one or more RNAs,” respectively.
As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.
Ranges recited herein include the defined boundary numerical values as well as sub-ranges encompassing any non-recited numerical values within the recited range. For example, a range from about 0.01 mM to about 10.0 mM includes both 0.01 mM and 10.0 mM. Non-recited numerical values within this exemplary recited range also contemplated include, for example, 0.05 mM, 0.10 mM, 0.20 mM, 0.51 mM, 1.0 mM, 1.75 mM, 2.5 mM 5.0 mM, 6.0 mM, 7.5 mM, 8.0 mM, 9.0 mM, and 9.9 mM, among others. Exemplary sub-ranges within this exemplary range include from about 0.01 mM to about 5.0 mM; from about 0.1 mM to about 2.5 mM; and from about 2.0 mM to about 6.0 mM, among others.
As used herein, the terms “regulation” and “modulation” may be utilized interchangeably and may include “promotion” and “induction.” For example, a transcription factor that regulates or modulates expression of a target gene may promote and/or induce expression of the target gene. In addition, the terms “regulation” and “modulation” may be utilized interchangeably and may include “inhibition” and “reduction.” For example, a transcription factor that regulates or modulates expression of a target gene may inhibit and/or reduce expression of the target gene.
As used herein, the term “sample” may include “biological samples” and “non-biological samples.” Biological samples may include samples obtained from a human or non-human subject. Biological samples may include but are not limited to, blood samples and blood product samples (e.g., serum or plasma), urine samples, saliva samples, fecal samples, perspiration samples, and tissue samples. Non-biological samples may include but are not limited to aqueous samples (e.g., watershed samples) and surface swab samples.
The term “target molecule” means any molecule of interest in a test sample and may include so-called “small molecules” or metabolites of small molecules. Target molecules may be referred to herein alternatively as “analytes,” “metabolites,” and “contaminants.” Exemplary target molecules include metabolites, chemical compounds, and nucleic acids. By way of example, but not by way of limitation, target molecules include phloroglucinol, mercury, arsenic or its oxides, nitrate, fluoride, cyanuric acid, lead, copper, zinc, chromium or its oxides, or atrazine.
The term “metabolite” means a molecule to which a target molecule is converted, for example, by one or more components such as enzymes that are present in a cell-free protein synthesis (CFPS) reaction mixture and/or that are added to a CFPS reaction mixture.
The term “transcription factor” refers to a protein that regulates transcription of another protein, typically by interacting by one or more cis-acting DNA sequence in or near the promoter for the other protein. A transcription factor may increase expression or decrease expression depending upon whether the transcription factor is activated or deactivated. A transcription factor may become activated or deactivated by an interaction with another molecule (e.g., a metabolite as described above). Such transcription factors are termed allosteric transcription factors.
The term “reporter molecule” refers to a molecule (e.g., a reporter protein or RNA) that can be detected in a reaction mixture, such as a CFPS reaction mixture, typically in response to the presence of a target molecule or a metabolite thereof being present in the reaction mixture. For example, a reporter molecule may be expressed and detected in a CFPS reaction mixture when a target molecule or a metabolite thereof activates a transcription factor which promotes expression of the reporter protein in the CFPS reaction mixture. Exemplary reporter molecules include fluorescent molecules, such as Green Fluorescent Protein and super-folded Green Fluorescent Protein. Any number of reporter molecules well known in the art (Yellow, Blue, and Red Fluorescent Proteins, mCherry, etc.) can be used in the methods, systems, compositions, and kits of the present disclosure.
The term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.
As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, bacteriophage polymerases such as, but not limited to, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.
As used herein, “expression template” refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably.
As used herein, “translation template” refers to an RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptide or protein.
As used herein, coupled transcription/translation (“Tx/Tl”), refers to the de novo synthesis of both RNA and a sequence defined biopolymer from the same extract. For example, coupled transcription/translation of a given sequence defined biopolymer can arise in an extract containing an expression template and a polymerase capable of generating a translation template from the expression template. Coupled transcription/translation can occur using a cognate expression template and polymerase from the organism used to prepare the extract. Coupled transcription/translation can also occur using exogenously-supplied expression template and polymerase from an orthogonal host organism different from the organism used to prepare the extract. In the case of an extract prepared from a yeast organism, an example of an exogenously-supplied expression template includes a translational open reading frame operably coupled a bacteriophage polymerase-specific promoter and an example of the polymerase from an orthogonal host organism includes the corresponding bacteriophage polymerase.
Polynucleotides and Uses Thereof
The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).
The terms “nucleic acid” and “oligonucleotide,” as used herein, may refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.
Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Letters 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, incorporated herein by reference.
Regarding polynucleotide sequences, the terms “percent identity” and “% identity” refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).
Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code where multiple codons may encode for a single amino acid. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. For example, polynucleotide sequences as contemplated herein may encode a protein and may be codon-optimized for expression in a particular host. In the art, codon usage frequency tables have been prepared for a number of host organisms including humans, mouse, rat, pig, E. coli, plants, and other host cells.
A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.
The term “amplification reaction” refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription, the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary “amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two-step cycles have a high temperature denaturation step followed by a hybridization/elongation (or ligation) step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.
The terms “target,” “target sequence,” “target region,” and “target nucleic acid,” as used herein, are synonymous and may refer to a region or sequence of a nucleic acid which is to be hybridized and/or bound by another nucleic acid.
The term “hybridization,” as used herein, refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).
The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.
Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. For example, primers may contain an additional nucleic acid sequence at the 5′ end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product, or which enables transcription of RNA (for example, by inclusion of a promoter) or translation of protein (for example, by inclusion of a 5′-UTR, such as an Internal Ribosome Entry Site (IRES) or a 3′-UTR element, such as a poly(A)n sequence, where n is in the range from about 20 to about 200). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.
As used herein, a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid. Typically, a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases. Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences that contain the target primer binding sites.
As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, RNA polymerases of bacteriophages (e.g. T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, Syn5 RNA polymerase), and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.
Also contemplated for us in the disclosed compositions, systems, kits, and methods are engineered RNA polymerase. For example, an engineered polymerase may be a non-naturally occurring RNA polymerase whose amino acid sequence has been engineered to include one or more of an insertion, a deletion, or a substitution relative to the amino acid sequence of a naturally occurring or wild-type RNA polymerase.
The term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.
As used herein, “an engineered transcription template” or “an engineered expression template” refers to a non-naturally occurring nucleic acid that serves as substrate for transcribing at least one RNA. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably. Engineered include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use in a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms.
“Transformation” or “transfection” describes a process by which exogenous nucleic acid (e.g., DNA or RNA) is introduced into a recipient cell. Transformation or transfection may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation or transfection is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection or non-viral delivery. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, electroporation, heat shock, particle bombardment, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The term “transformed cells” or “transfected cells” includes stably transformed or transfected cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed or transfected cells which express the inserted DNA or RNA for limited periods of time.
The polynucleotide sequences contemplated herein may be present in expression vectors. For example, the vectors may comprise a polynucleotide encoding an ORF of a protein operably linked to a promoter. “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. Vectors contemplated herein may comprise a heterologous promoter operably linked to a polynucleotide that encodes a protein. A “heterologous promoter” refers to a promoter that is not the native or endogenous promoter for the protein or RNA that is being expressed.
As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or another RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.”
The term “vector” refers to some means by which nucleic acid (e.g., DNA) can be introduced into a host organism or host tissue. There are various types of vectors including plasmid vector, bacteriophage vectors, cosmid vectors, bacterial vectors, and viral vectors. As used herein, a “vector” may refer to a recombinant nucleic acid that has been engineered to express a heterologous polypeptide (e.g., the fusion proteins disclosed herein). The recombinant nucleic acid typically includes cis-acting elements for expression of the heterologous polypeptide.
In the methods contemplated herein, a host cell may be transiently or non-transiently transfected (i.e., stably transfected) with one or more vectors described herein. A cell transfected with one or more vectors described herein may be used to establish a new cell line comprising one or more vector-derived sequences. In the methods contemplated herein, a cell may be transiently transfected with the components of a system as described herein (such as by transient transfection of one or more vectors), and modified through the activity of a complex, in order to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
Peptides, Polypeptides, and Proteins
As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeable to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.
A “protein” as contemplated herein typically comprises a polymer of naturally or non-naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation), hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).
The proteins disclosed herein may include “wild type” proteins and variants, mutants, and derivatives thereof. As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein, a “variant, “mutant,” or “derivative” refers to a protein molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, a mutant or variant molecule may have one or more insertions, deletions, or substitution of at least one amino acid residue relative to a reference polypeptide.
Regarding proteins, a “deletion” refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues. A deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, or more amino acids residues. A deletion may include an internal deletion and/or a terminal deletion (e.g., an N-terminal truncation, a C-terminal truncation or both of a reference polypeptide). A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a deletion relative to the reference polypeptide sequence.
Regarding proteins, “fragment” is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full-length polypeptide. A fragment may include an N-terminal truncation, a C-terminal truncation, or both truncations relative to the full-length protein. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence.
Regarding proteins, the words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include an insertion or addition relative to the reference polypeptide sequence. A variant of a protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions.
Regarding proteins, the phrases “percent identity” and “% identity,” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
Regarding proteins, percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Regarding proteins, the amino acid sequences of variants, mutants, or derivatives as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative protein may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. The following table provides a list of exemplary conservative amino acid substitutions which are contemplated herein:
Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. Non-conservative amino acids typically disrupt (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
The disclosed proteins, mutants, variants, or described herein may have one or more functional or biological activities exhibited by a reference polypeptide (e.g., one or more functional or biological activities exhibited by wild-type protein).
In some embodiments of the disclosed compositions, systems, kits, and methods, the components may be substantially isolated or purified. The term “substantially isolated or purified” refers to components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
Cell-Free Protein Synthesis (CFPS)
The disclosed subject matter relates in part to methods, devices, kits and components for cell-free protein synthesis. Cell-free protein synthesis (CFPS) is known and has been described in the art. (See, e.g., U.S. Pat. Nos. 6,548,276; 7,186,525; 8,734,856; 7,235,382; 7,273,615; 7,008,651; 6,994,986; 7,312,049; 7,776,535; 7,817,794; 8,298,759; 8,715,958; 9,005,920; U.S. Publication No. 2014/0349353, U.S. Publication No. 2016/0060301, U.S. Publication No. 2018/0016612, and U.S. Publication No. 2018/0016614, the contents of which are incorporated herein by reference in their entireties). A “CFPS reaction mixture” typically contains a crude or partially-purified bacterial extract (as used herein the terms “extract” and “lysate” are used interchangeably), an RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template. In some aspects, the CFPS reaction mixture can include exogenous RNA translation template. In other aspects, the CFPS reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase. In these other aspects, the CFPS reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In these other aspects, additional NTP's and divalent cation cofactor can be included in the CFPS reaction mixture. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention. For example, the cellular transcription and translational machinery may be provided in a lysate from an engineered bacterial strain, or the transcription and translational machinery may be purified separately and reconstituted to defined concentrations. In some embodiments, a lysate may be from an engineered bacterial strain, and include cellular transcriptional and translational machinery, and may also include other as other cellular proteins.
The disclosed cell-free protein synthesis systems may utilize components that are crude and/or that are at least partially isolated and/or purified. As used herein, the term “crude” may mean components obtained by disrupting and lysing cells and, at best, minimally purifying the crude components from the disrupted and lysed cells, for example by centrifuging the disrupted and lysed cells and collecting the crude components from the supernatant and/or pellet after centrifugation. The term “isolated or purified” refers to components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
An aspect of the invention is a platform for preparing a sequence defined protein in vitro which may be utilized for detecting a target molecule or metabolite thereof. The platform for preparing a sequence defined polymer or protein in vitro comprises a cellular extract from a host strain. Because CFPS exploits an ensemble of catalytic proteins prepared from the crude lysate of cells, the cell extract (whose composition is sensitive to growth media, lysis method, and processing conditions) is the most critical component of extract-based CFPS reactions. A variety of methods exist for preparing an extract competent for cell-free protein synthesis, including U.S. patent application Ser. No. 14/213,390 to Michael C. Jewett et al., entitled METHODS FOR CELL-FREE PROTEIN SYNTHESIS, filed Mar. 14, 2014, and now published as U.S. Patent Application Publication No. 2014/0295492 on Oct. 2, 2014, and U.S. patent application Ser. No. 14/840,249 to Michael C. Jewett et al., entitled METHODS FOR IMPROVED IN VITRO PROTEIN SYNTHESIS WITH PROTEINS CONTAINING NON STANDARD AMINO ACIDS, filed Aug. 31, 2015, and now published as U.S. Patent Application Publication No. 2016/0060301, on Mar. 3, 2016, the contents of which are incorporated by reference.
The platform may comprise an expression template, a translation template, or both an expression template and a translation template. The expression template serves as a substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). The translation template is an RNA product that can be used by ribosomes to synthesize the sequence defined biopolymer. In certain embodiments the platform comprises both the expression template and the translation template. In certain specific embodiments, the platform may be a coupled transcription/translation (“Tx/Tl”) system where synthesis of translation template and a sequence defined biopolymer from the same cellular extract.
The platform may comprise one or more polymerases capable of generating a translation template from an expression template. The polymerase may be supplied exogenously or may be supplied from the organism used to prepare the extract. In certain specific embodiments, the polymerase is expressed from a plasmid present in the organism used to prepare the extract and/or an integration site in the genome of the organism used to prepare the extract.
Altering the physicochemical environment of the CFPS reaction to better mimic the cytoplasm can improve protein synthesis activity. The following parameters can be considered alone or in combination with one or more other components to improve robust CFPS reaction platforms based upon crude cellular extracts (for examples, S12, S30 and S60 extracts).
The temperature may be any temperature suitable for CFPS. Temperature may be in the general range from about 10° C. to about 40° C., including intermediate specific ranges within this general range, include from about 15° C. to about 35° C., from about 15° C. to about 30° C., form about 15° C. to about 25° C. In certain aspects, the reaction temperature can be about 15° C. about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C.
The CFPS reaction can include any organic anion suitable for CFPS. In certain aspects, the organic anions can be glutamate, acetate, among others. In certain aspects, the concentration for the organic anions is independently in the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as about 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM and about 200 mM, among others.
The CFPS reaction can also include any halide anion suitable for CFPS. In certain aspects the halide anion can be chloride, bromide, iodide, among others. A preferred halide anion is chloride. Generally, the concentration of halide anions, if present in the reaction, is within the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as those disclosed for organic anions generally herein.
The CFPS reaction may also include any organic cation suitable for CFPS. In certain aspects, the organic cation can be a polyamine, such as spermidine or putrescine, among others. Preferably polyamines are present in the CFPS reaction. In certain aspects, the concentration of organic cations in the reaction can be in the general about 0 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 2 mM. In certain aspects, more than one organic cation can be present.
The CFPS reaction can include any inorganic cation suitable for CFPS. For example, suitable inorganic cations can include monovalent cations, such as sodium, potassium, lithium, among others; and divalent cations, such as magnesium, calcium, manganese, among others. In certain aspects, the inorganic cation is magnesium. In such aspects, the magnesium concentration can be within the general range from about 1 mM to about 50 mM, including intermediate specific values within this general range, such as about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, among others. In preferred aspects, the concentration of inorganic cations can be within the specific range from about 4 mM to about 9 mM and more preferably, within the range from about 5 mM to about 7 mM.
The CFPS reaction includes NTPs. In certain aspects, the reaction use ATP, GTP, CTP, and UTP. In certain aspects, the concentration of individual NTPs is within the range from about 0.1 mM to about 2 mM.
The CFPS reaction can also include any alcohol suitable for CFPS. In certain aspects, the alcohol may be a polyol, and more specifically glycerol. In certain aspects the alcohol is between the general range from about 0% (v/v) to about 25% (v/v), including specific intermediate values of about 5% (v/v), about 10% (v/v) and about 15% (v/v), and about 20% (v/v), among others.
Biosensors: Compositions, Kits, and Systems
The technology described herein relates generally to microbial-based biosensor compositions, systems, and kits for the detection of small molecules and analytes (e.g., metabolites, chemical compounds, nucleic acids), based on an analyte-responsive transcription factor-DNA binding mechanism, resulting in the expression of a detectable reporter protein. The biosensors employ one or more analyte-responsive transcription factor-DNA binding platforms for the cell free detection of target molecules. In some embodiments, the biosensor systems include one or more signal amplifiers to provide a cascade of polymerase expression, and/or include one or more inhibitors to decrease background (increase signal to noise ratio) of the reporter protein.
As used herein, the term “biosensor” refers to a reaction mixture comprising all of the components necessary to detect an analyte of interest. In some embodiments, the biosensor is provided as freeze-dried components contained in a vessel, such as a microfuge tube, test tube, or a multi-well plate. Upon rehydration of the components with a liquid or liquefied sample, the analyte of interest, if present, initiates a reaction in the vessel resulting in the expression of a reporter molecule.
The biosensor is designed to detect a ligand (the analyte) of an allosteric transcription factor. Once the ligand binds its transcription factor, a cascade of transcription and translation events occurs. Accordingly, the biosensors comprise the components for transcription and translation reactions and include the necessary enzymes, co-factors, nucleotides, amino acids, energy source, etc. These components can be provided individually, or can be provided as one or more extracts for CFPS as described above.
By way of example, a biosensor of the present disclosure comprises one or more lysates from engineered bacterial strains, the lysate comprising cellular transcriptional and translational machinery, and optionally other cellular proteins, co-factors, energy sources (e.g., ATP-based cellular energy or non-phosphate based energy); a biosensor molecule that modulates the expression of a target DNA sequence in a DNA transcription template (e.g., an ATF); a DNA transcription template whose expression is configured to be regulated by the biosensor molecule, and encoding the expression of additional RNA polymerase not present in the lysate (e.g., an exogenous or orthogonal RNA polymerase); and a second DNA transcription template encoding the expression of a reporter molecule (e.g., a reporter protein or RNA molecule) whose transcription is controlled by the expressed additional RNA polymerase (e.g., wherein the second DNA transcription template comprises a promoter for the exogenous or orthogonal RNA polymerase).
The biosensors comprise at least one biosensor molecule, such as an allosteric transcription factor (ATF). The term “allosteric transcription factor” as used herein refers to regulatory proteins that contain a DNA-binding domain as well as a ligand-binding domain that is able to recognize small molecules with high specificity and selectivity. In the presence of a target small molecule (i.e., the transcription factor ligand), transcription factor affinity for its DNA binding sequence is modulated, facilitating the repressor or derepressor regulation of downstream gene expression. In some embodiments, the biosensors disclosed herein comprise a plasmid (e.g., a sensor plasmid) that expresses the ATF either in the biosensor (e.g., transcription and translation of the ATF occurs upon rehydration of the biosensor components by adding liquid the sample), or in the host strain used for making the biosensor (e.g., as a component of a CFPS reaction). For example, in some embodiments, the biosensors comprise the ATF protein, and the biosensor molecule is overexpressed in the host strain prior to making the extract for cell-free protein synthesis. Additionally or alternatively, the biosensor molecule (e.g., an ATF) comprises an isolated protein and is added to the biosensor components.
As used herein, the term “sensor plasmid” refers to a plasmid comprising a promoter which drives the expression of an allosteric transcription factor. See e.g.,
The biosensor disclosed herein also comprise a reporter plasmid, or a linear reporter DNA construct. The reporter molecule can be any detectable protein. In some embodiments, the reporter protein can be visualized without the use of additional equipment or reagents. While GFP and sfGFP are exemplified herein, the biosensors are not intended to be so limited, and any number of detectable protein markers can be employed and include, but are not limited to green fluorescent protein, red fluorescent protein, blue fluorescent protein or any derivatives thereof. In some embodiments, the reporter comprises a den enzyme that produces a visible signal, such as catechol 2,3-dioxygenase (C23D0) beta-galactosidase (LacZ), or glucuronidase (GusA).
The reporter plasmid or linear construct also comprises a promoter to drive the expression of the reporter molecule. The promoter may be reactive to the ATF and its polymerase (e.g., as shown in
The biosensors disclosed herein may also include one or more signal amplification constructs in the form of plasmid or linear DNA constructs (see e.g.,
In the scheme outlined in
By way of example, allosteric transcription factors that are activated or deactivated by an interaction with another molecule include those shown below in Table 1. Their promoter sequences are also provided.
As described above, the promoter sequence responsive to the activated ATF drives the expression of a reporter molecule or an orthogonal polymerase. In some embodiments, the promoter sequence responsive to the activated ATF comprises an E. coli promoter sequence. By way of example, variants of the E. coli J23119 promoter sequence is used. Plasmids were assembled using isothermal (Gibson) assembly and confirmed by Sanger sequencing. The sequences for the ArsR (#78635) and MerR (#123148) genes were obtained from Addgene from Dr. Baojun Wang's lab. The sequences for the NarX and NarL plasmids were a generous gift from Dr. Jeffrey Tabor's lab. Other constitutive promoters engineered to have aTF-binding sites (e.g., operators grafted into or after the entire Anderson promoter collection in the BioBricks catalog) would be appropriate. The promoter can essentially be any promoter, so long as it is responsive to the selected ATF and the biosensor includes an appropriately matched polymerase.
Orthogonal polymerases and matched promoters can be introduced in the biosensors to generate a cascade of polymerase transcription and translation (see e.g.,
CTCACTAT
CTCACTAT
GACACTAT
ATCACTAT
GTCACTAT
By way of example but not by way of limitation, expression vectors that can be used in the methods and systems disclosed herein are provided in Table 3 below.
The biosensors disclosed herein may be multiplexed; that is, more than one target can be detected in a single reaction vessel. By way of example, as shown in
In some embodiments, the biosensors are optimized, e.g., to provide detectable signals in a shorter time, and/or cleaner signal (e.g., with less background). Cascaded systems, as described above, provide one means of optimization. Another means of optimization includes regulating the T7 polymerase activity and/or expression. As shown in
In some embodiments, optimization includes “additional” RNA polymerases that have been specifically evolved or engineered for specificity for only a single promoter to avoid crosstalk.
In some embodiments, optimization includes reporter protein comprising orthogonal fluorescence or absorbance spectra, or catalyze enzymatic reactions that produce different colors.
In some embodiments, the target molecule to be detected comprises one or more of phloroglucinol, mercury, arsenic or its oxides, nitrate, fluoride, cyanuric acid, lead, copper, zinc, chromium or its oxides or atrazine. In some embodiments, the target molecule to be detected comprises RNA or DNA.
In some embodiments, the nucleic acids provided as components of the biosensor are amplified using an isothermal strategy prior to sensor activation. By way of example, nucleic acid sequence-based amplification (NASBA) and recombinant polymerase amplification (RPA) may be used.
Methods employing the biosensors are also contemplated herein. For example, methods of detecting a target molecule (e.g., a metabolite, a chemical compound, a nucleic acid) in a biological or environmental sample may include: (i) obtaining a biological or environmental sample which may or may not contain the target molecule and optionally concentrating and/or solubilizing the target molecule in the sample if necessary; (ii) adding the sample and/or the optionally concentrated and/or solubilized target molecule in the sample to a cell-free protein synthesis (CFPS) reaction, wherein if the target molecule is present in the sample then an output is generated (e.g., a visual, electronic, or optical output); wherein the output is generated via steps that include: (i) the target molecule inducing expression of an RNA polymerase from a first DNA transcription template, wherein the expressed RNA polymerase is not present in the CFPS reaction prior to its expression, optionally wherein the expression of the RNA polymerase is induced via a biosensor molecule in the presence of the target molecule; (ii) the expressed RNA polymerase expresses a reporter molecule from a second DNA transcription template (e.g., wherein the second DNA transcription template comprises a promoter for the expressed RNA polymerase) and the reporter molecule generates an output either directly or indirectly.
Applications and Advantages
Applications of the disclosed technology may include but are not limited to: (i) improving the sensitivity of molecular diagnostics, such as field-deployable molecular diagnostics; (ii) improving the maximum detectable signal of molecular diagnostics; (iii) improving the response transfer curve of molecular diagnostics for more sensitive and sigmoidal switching behavior; and (iv) enabling one-pot multiplexing of several cell-free sensors.
Advantages of the disclosed technology may include but are not limited to: (i) the development of sensors having an improved limit of detection for arbitrary analytes (target molecules) which is enhanced compared to a no-signal amplification condition, as well as the reporter signal in the ON (i.e. target chemical present) state; (ii) the development of sensors having a response which is more “switchlike”, or sigmoidal, enabling better semi-quantitative determination of concerning concentrations of relevant analytes; and (iii) the development of sensors which are extremely modular and adapted to various reporter outputs, which also enables one-pot multiplexing of various detection schemes with different fluorescent proteins or enzymes. Point-of-care, field-deployable diagnostics could allow consumers to rapidly and inexpensively determine water quality, be used for personalized health monitoring, be used for point-of-use health monitoring.
The examples provided herein are not intended to be limiting, and are provided to demonstrate aspects of the present technology.
In general, a cascaded and noncascaded sensor requires three plasmids that are designed and assembled using standard molecular biology strategies (isothermal assembly, restriction cloning, blunt-end ligation, solid-phase oligonucleotide synthesis, etc.). One plasmid encodes the target allosteric transcription factor (e.g., CueR) under the control of the wild-type T7 RNAP promoter. The other two encode the responsive promoter sequence (e.g., pCue) upstream of a reporter, either the sfGFP coding sequence (the noncascaded sensor) or T7 RNAP (the cascaded sensor). The natural promoter sequences are typically used, although for promoters derived from non-E. coli hosts, mutations to the consensus −10 and −35 sites for sigma-70 promoters (TTGACA, TATAAT) can be helpful to generate stronger promoters that are still functionally regulated. Multiple operator sites can also be placed in tandem in a promoter to improve the ability of the aTF to regulate gene expression. The same cascaded reporter plasmid (e.g., pAKSIRV-sfGFP) is used for all experiments described herein.
The optimized reporter plasmids are isolated and sequence-confirmed and the reporter plasmids are purified to high concentration by midiprep. The plasmid encoding the aTF is transformed into a protein expression strain of E. coli (e.g., BL21 (DE3) or its derivatives) and an extract is prepared using previously reported protocols (e.g., citation 18). Separately, a “blank” extract is prepared from the base protein production strain. Cell-free extracts for transcriptional sensing are typically prepared by lysis and post-lysis clarification including ribosomal runoff reaction and dialysis, followed by aliquoting and flash-freezing on liquid nitrogen.
A series of tuning experiments are next used to optimize the sensor. First, the optimal concentration of the aTF (CueR) is found for the non-cascaded sensor (e.g., pCue-sfGFP) through a series of ratiometric titrations between the aTF-enriched extract and the blank extract in both the presence and absence of analyte (see e.g., the data in
Next, the optimal ratio of aTF enriched- and blank extract is used to optimize the concentration of the sensor plasmid sequence for the cascade (e.g., pCue-AKSIRV) (see e.g., the data in
The innovations disclosed herein, include but are not limited to: (i) deploying a cell-free sensor that produces a bacteriophage RNA polymerase in the presence of a target chemical or nucleic acid; (ii) catalytic amplification of that bacteriophage polymerase with a positive feedback template, and co-expression of a reporter protein from the bacteriophage polymerase's cognate promoter, in one pot; and (iii) deploying multiple engineered polymerase variants in a single pot which allows for multiplexed detection of several analytes at once. The disclosed innovations allow for overall improved signal of the sensor and also makes the sensor be more switchlike, greatly increase the limit of detection of the disclosed sensors; and allow for practical sensing of several contaminants using a single reaction, which simplifies the device and decreases overall cost. The exemplary models and designs presented herein uniquely demonstrated the ability of the cascaded amplifiers to be applied to and monitor lead, mercury, nitrate, cadmium, copper, fluoride, chromate, and arsenic.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
U.S. Pat. Nos.: U.S. Pat. Nos. 5,478,730; 5,556,769; 5,665,563; 6,168,931; 6,518,058; 6,783,957; 6,869,774; 6,994,986; 7,118,883; 7,189,528; 7,338,789; 7,387,884; 7,399,610; 8,357,529; 8,574,880; 8,703,471; 8,999,668; 9,410,170; and US952813; the contents of which are incorporated herein by reference in their entirety.
U.S. Patent Publications: US20040209321; US20050170452; US20060211085; US20060234345; US20060252672; US20060257399; US20060286637; US20070026485; US20070154983; US20070178551; US20080138857; US20140295492; US20160060301; US20180016612; US20180016614; US20160312312; and US20160362708; the contents of which are incorporated herein by reference in their entirety.
Published International Applications: WO2003056914A1; WO2004013151A2; WO2004035605A2; WO2006102652A2; WO2006119987A2; WO2007120932A2; WO2014144583; and WO2017117539; the contents of which are incorporated herein by reference in their entirety.
In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
This application is a Continuation-In-Part of International Application PCT/US2020/063133, filed Dec. 3, 2020, which claims the benefit of U.S. Provisional Application No. 62/943,094 filed on Dec. 3, 2019, and U.S. Provisional Application No. 63/003,724 filed on Apr. 1, 2020, the contents of which are incorporated herein by reference in their entireties.
This invention was made with government support under FA8650-15-2-5518 awarded by the Department of Defense, Air Force Research Laboratory. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62943094 | Dec 2019 | US | |
| 63003724 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2020/063133 | Dec 2020 | US |
| Child | 17131538 | US |
